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Tuesday, June 26, 2007

Alnylam Pharmaceuticals, arguably the leading company in RNAi Therapeutics, today announced the start of phase II clinical studies for ALN-RSV01, an siRNA for the treatment of Respiratory Syncytial Virus (RSV) infection. RSV infection is the leading cause of infant hospitalization in the US and a significant risk factor for the immune-compromised and elderly. Although a neutralising antibody exist for the prevention of RSV infection, no drug has been shown so far to be effective in the treatment of RSV infection.

Today marks another milestone in the rapid, but at the same time circumspect development of ALN-RSV01 from the test tube to the clinic. It demonstrates just how quickly it is possible to develop RNAi as antivirals. This is because the viral sequence alone allows us to start designing and testing siRNAs for their antiviral activity. It is not surprising therefore that the NIH and Department of Defense is interested in fostering this technology for the fight against bioterrorism and pandemic flu. Indeed, Alnylam may leverage their experience in RSV and translate it into their pandemic flu program for which an IND is planned by the end of the year. Another company, Nastech, of Bothell, Washington, is also in the discovery phase of an RNAi therapeutic for pandemic flu, as was Sirna Therapeutics before their acquisition by Merck.

Alnylam’s first phase II study is an experimental challenge study in which volunteers are nasally infected with an attenuated strain of RSV and ALN-RSV01 given either before or after infection. The goal of this randomised 90 patient double-blind, placebo controlled study is to first establish safety, but more importantly antiviral efficacy as measured by incidence of infection, viral titer, and symptomatic differences. If successful, this would constitute human proof-of-concept of a human RNAi Therapeutic and represent another major de-risking event on the path to establishing RNAi as a whole new platform for innovative drugs.

PS: In another RNAi pipeline event, Intradigm, an RNAi delivery company based in Palo Alto, California, announced today that they have officially selected ICS-283 as a development program for cancer. ICS-283 is designed to target cancer angiogenesis and phase I studies are expected to start in 2008.

Wednesday, June 20, 2007

One often cited challenge for the wide application of RNAi in the treatment of disease is the systemic delivery of the RNAi inducing agent to the appropriate target cells. Systemic delivery is the ability to deliver RNAi to target cells following intravenous or even oral administration, whereas local, also called “Direct RNAi” approaches for eye, lung, and mucosal diseases appear to be less of a challenge.

Neurodegenerative diseases and other brain-related diseases are an attractive area for the application of an RNAi therapeutic, due to significant unmet medical needs and the existence of well validated gene targets. The delivery of RNAi to the CNS, however, is challenging due to the presence of the blood-brain barried (BBB) which makes it difficult for macromolecules to exit blood vessels and enter the CNS. First generation RNAi therapeutics for the CNS may therefore rely on direct injection or device implantation as is illustrated by the current Alnylam-Medtronic collaboration. Systemic delivery, however, would have the added advantage that a therapeutic would be well distributed throughout the brain due to the extensive vascularisation of the brain. Local administration strategies often suffer from the fact that the therapeutic agent is restricted to the site of application.

The paper published this Monday online in the leading scientific journal Nature by Kumar et al. shows that the ability of the rabies virus to cross the BBB can be conferred onto an siRNA if it was non-covalently linked to a 29 amino acid peptide from the rabies virus. Although the mechanism by which this peptide achieves this is unclear, the authors show that once in the CNS it binds to acetylcholine-receptors on neuronal cells and concomitantly delivers the siRNA load to those cells. A number of endogenously expressed genes were thereby targeted achieving 30-70% knockdown efficiencies, including a 70% reduction in SOD-1 activity.

SOD-1 when mutated is linked to the debilitating neuronal disease ALS (Amyotrophic Lateral Sclerosis; aka Lou Gehrig’s) for which there is no real treatment. Interestingly, preclinical therapeutic effects have previously been achieved with lentivirally delivered shRNAs. It is therefore hoped that ultimately RNAi may offer the first treatment alternative for ALS that addresses the underlying cause of the disease.

In another impressive demonstration of the system, almost all mice infected with an otherwise fatal dose of Japanese Encephalitis Virus were rescued when they received the rabies-peptide siRNA combination. This follows a report last year by the same group that single siRNAs could also protect against the related West Nile Virus.

This report demonstrates that progress in the delivery of RNAi Therapeutics can come from unexpected sources and illustrates the force and commitment with which this technology is moving forward. It is reasonable to assume that one by one, similar solutions and improvements to the RNAi delivery challenge will come for other difficult-to-target tissues. Further improvements to the present system should also be possible, as the authors suggest, for example through covalent conjugation of the siRNA to the peptide to avoid premature dissociation of the siRNA from the rabies peptide. It is also tempting to imagine that high-throughput approaches such as one involving peptide phage display libraries could yield siRNA-peptide combinations for other tissues of the body.

The key will be that the results can be reproduced in another laboratory, is easy-to-use, and similarly non-immunogenic in man as it is in mice. In that case, I would not be surprised to see the technology being licensed by a major RNAi Therapeutics company. The proximity of Harvard to Alnylam’s Cambridge, Mass., headquarters certainly makes this company a prime candidate for that. It should also be noted that one of the co-authors, Beverly Davidson from the University of Iowa, an acknowledged RNAi expert with experience in brain-related RNAi applications, had been associated with Sirna Therapeutics prior to their acquisition by Merck.

Sunday, June 17, 2007

Today, I will pause and take stock of what I think have been remarkable months for the public understanding of RNAi Therapeutics.

Indeed, the climate for RNAi Therapeutics has never been better. Only recently, a number of external factors have come together that should significantly support the progress of developing the basic science of RNAi into a whole new drug class. Following the award of the Nobel Prize for the discovery of RNAi late last year, the political willingness to support RNAi has begun to extend beyond the NIH to initiatives like the one announced last month by the Governor of Massachusetts to provide $1 billion support over the next 10 years for innovative life sciences, particularly therapeutic RNAi.

These developments have consequently reached a stage where the leading political/financial journal The Economist is featuring SMALL RNAs on this week’s title page of the print issue, under the heading of “Biology’s Big Bang- Unravelling the secrets of RNA” (http://www.economist.com/printedition/). All this excitement comes at a time when the pace of Investigational New Drug applications for RNAi Therapeutics has reached one IND filed every month such that eight announced RNAi Therapeutic clinical development programs are now underway in the US and Europe. And there is every sign that this pace will pick up before it slows down.

Appropriately, the second half of this year has the potential to mark another milestone in the validation of RNAi as a human therapeutic. The leading RNAi company Alnylam intends to announce by the end of this month the detailed clinical plan for a phase II experimental infection study of ALN-RSV01, an siRNA for the treatment of RSV infection in infants, the elderly and other immune-compromised adults. In this study, volunteers are inoculated nasally with attenuated RSV and the effect of siRNA on various parameters of infection is measured, most importantly viral titers.

According to the company, the study will involve 90 volunteers in a randomised, double-blind, placebo-controlled trial. If results are as hoped, this trial therefore has the potential to show for the first time statistically significant antiviral, that is therapeutic activity of an RNAi agent in man. This would represent another major de-risking event which should not only benefit Alny’s corporate objectives, but the field as a whole.

Note: Last week Alnylam (ALNY) gained almost 10% to $16.89, possibly in anticipation of these potentially value-creating events. This comes after Alnylam lost 1/3 of its value from its all-time high of $24.46, partly pressured by short sale tactics following the issuance of new shares. It should be noted that little more than 11% is held non-institutionally and if the above events should attract new major investors, prices could easily be propelled higher. Notably, last Friday saw a number a large blocks changing hands on more than twice the average daily volume, sending the stock higher by 6.43% in one day.

Thursday, June 14, 2007

After some delay, Benitec and their collaborators from the City of Hope finally announced that they obtained regulatory approval to start phase I clinical trials for an RNA-based HIV gene therapy antiviral. The delay was caused by Benitec’s uncertain corporate future and difficulties generating sufficient amounts of clinical-grade lentiviral vectors, but scientifically the risk-(potential) benefit profile of these studies are promising.

This is the 8th RNAi clinical program and the second involving DNA-directed shRNAs. The gene therapy agent is a lentiviral vector expressing 3 different RNA molecules, each designed to interfere with a different aspect of HIV replication, each through a distinct mechanism of action. The plan is to immunise CD34+ hematopoietic progenitor cells and thereby all their progenies, including T-cells, with such vectors ex vivo and then re-administer the cells to the patients.

The RNAi portion is a U6-driven hairpin RNA targeting the tat/rev mRNA by RNAi. A nucleolar-localised TAR decoy RNA, also under the direction of a U6 promoter, is designed to mimic the HIV TAR element, thereby diverting TAR-binding factors by mimicry. Finally, a ribozyme against the CCR5 mRNA, a co-receptor for HIV infection, complements the 3-pronged approach. This approach is inspired by the current HAART HIV treatment paradigm where a cocktail of antiretroviral drugs has proven to be highly effective in suppressing HIV replication with only slow development of drug resistance.

In fact, the RNA-based vector which has been shown in tissue-culture experiments to be considerably active in inhibiting HIV replication may be synergistic with present therapies due to their unique mechanism of action, although non RNA-based anti-CCR5 treatments are currently developed by other companies as well. While Benitec used to pursue a triple RNAi approach for the treatment of HCV (a program now owned by Tacere), the present strategy may be advantageous since it is known that co-transcribed shRNAs or co-transfected siRNAs may compete with each other for cellular RNAi factors.

Ultimately, I expect that long-term expression of ideally all 3 RNAs will be important to confer a survival advantage onto the lentivirally transduced CD34+-derived cells. However, even if expression should be silenced eventually, the treatment is likely to give AIDS patients some reprieve. The study population will be 5 AIDS-related leukaemia patients from which CD34+ will be enriched from blood by apheresis, genetically modified, and then returned to the donor patient.

Given that this is a gene therapy with a vector that hasn’t been used in the clinic before, it is expected that this therapy will be used in AIDS/Lymphoma patients who are no more responsive to conventional treatments. Nevertheless, expect to hear results from this promising phase I trial within a year.

Wednesday, June 13, 2007

Before RNAi, there was antisense. Antisense strategies typically aim to suppress gene expression by either preventing translation initiation or degrading the messenger mRNA through an RNase H mechanism. As more mechanistic insight into the RNAi pathway was gained, it became clear that the final active, so called “guide” RNA that recognises and causes cleavage of the target RNA by RNAi is a single-stranded 19-23nt RNA bound to the catalytic RiSC endonuclease Argonaute 2 (in humans).

Sure enough, the provision of single-stranded RNAs was then shown by the Tuschl lab to be sufficient for inducing RNAi (Martinez et. al., 2002), although considerably higher concentrations were needed compared to the conventional double-stranded siRNAs in vitro. This work and potential applications thereof are the subject of a USPTO patent application by the Max Planck Society.

Nevertheless, ISIS Pharmaceuticals would claim that its antisense patents cover any therapy making use of a single-strand antisense mechanism, and in their interpretation this includes RNAi with single-stranded RNAs. ISIS’ leadership in nucleic acid technology such as nucleic acid modification and manufacturing technology is without doubt. This is also the reason why Alnylam, the leading RNAi company (of which Tuschl is a co-founder), has taken an exclusive license to a number of ISIS’ patents for the development of RNAi therapeutics, particularly for the use of modified siRNAs.

Although in tissue culture it is clear that double-stranded siRNAs are much more potent inducers of RNAi than single-stranded small RNAs, single-stranded RNAi molecules may have some utility for in vivo applications. In addition to the potential cost of goods advantage of a single-stranded versus a double-stranded approach, the delivery of single-strand RNA-like molecules to organs such as the liver may not even require as much as a formulation into a special delivery composition, as is suggested by the ability of single-stranded antisense compounds such as ISIS’ ApoB100-targeting 301012 to enter liver cells following subcutaneous injection. It is also possible, although not proven, that some of ISIS’ antisense compounds, particularly those with limited 5’ modifications, actually work through an RNAi mechanism.

The issue therefore relates to the value of a claim to a mechanism that was not even known to exist at the time when ISIS applied for patents relating to RNase-mediated gene suppression by an antisense mechanism involving ANY RNAse activity. While I think it is almost impossible that these claims will supersede the fundamental Fire-Mello and Tuschl II RNAi patents, they will probably carry some weight for the use of single-stranded RNAi molecules and ISIS is indeed pursuing such single-strand RNAi molecules for therapeutic purposes. Importantly, however, their utility in an in vivo setting still remains to be demonstrated.

Friday, June 8, 2007

170 million people worldwide are infected with the Hepatitis C virus. Hep C, an RNA virus, is emerging as a significant public health problem that is only set to grow exponentially as many chronic carriers are about to develop liver cirrhosis and cancer after having been infected for a number of years.

Currently, treatment options for Hep C are essentially limited to a combination of interferon and ribavirin, both antivirals of which the mechanism of action is not completely understood. However, the need for additional treatment options has never been greater. While about 50% of those who adhere to treatment will be “cured” of the virus, many that start therapy do not complete the full treatment regimen due to the sometimes severe side-effect profile and the need for prolonged treatment.

The good news is that there is a whole generation of promising new drugs currently in pre-clinical and clinical development that target the virus directly. The most advanced of these are small molecule inhibitors of the Hep C protease and polymerase, some of which are about to enter phase III clinical trials. Early experience with Vertex Pharmaceutical’s VX-950 e.g. has shown impressive response rates of around 80-90% and reductions in viral loads and is expected to become an important part of future treatment strategies, mostly likely involving combination therapy due to viral resistance. Nevertheless, some of these drugs did not prove efficacious or safe and were discontinued. Morever, those drugs that will get approval will not work in every patient due to variability of the genetic background of the host (=patient) and the virus, and still more treatment options are desirable.

RNAi, through its ability to target the RNA genome of the virus itself, offers a unique opportunity in helping close that gap further. Its unique mechanism of action should contribute to shortening the course of treatment and enhancing response rates. Curiously, Hep C’s cousin, the Hepatitis B virus, was the first virus for which in vivo efficacy of RNAi was demonstrated. This is because of a lack of a good animal model system for Hep C that led a number of groups to target Hep B as a model system for Hep C RNAi treatments as both viruses share the same host tissue and delivery is considered as the main hurdle to achieve treatment success of RNAi. Despite the early promise, corporate actions, however, now seem to have put Hep C RNAi on the backburner.

Before being aquired by Merck, Sirna Therapeutics with the help of Protiva scientists demonstrated promising knockdown of Hep B in mice using the SNALP-siRNA delivery system. They even presented non-human primate Hep C data during corporate presentations and claimed treatment success in a limited number of animals. Progress, however, seems to have stalled as a result of a legal dispute surrounding the SNALP delivery system. Another sorry example is Benitec which had plans to progress a Hep C program, most likely involving a cocktail of DNA-directed shRNAs, to the clinic this year. As funding ran out and the company was forced to move back to Australia, management decided to spin off the Hep C program to a new company, Tacere, which, also short of money, is now struggling to extract any value out of the Hep C program.

It can only be hoped that the potential of RNAi for Hep C is not forgotten. Certainly, qualified academicians, clinicians, RNAi delivery systems, and RNAi targets are all there and waiting. The trick is to bring them together and give them funding to get on with what they are best at.

Monday, June 4, 2007

The most widely known and used triggers of RNAi are small interfering RNAs (siRNAs) and DNA-directed hairpin structures. Whereas siRNAs are channelled into the downstream steps of the RNAi pathway, the RiSC complex, hairpin RNAs in humans first have to undergo processing by the endogenous microRNA pathway to yield the active small silencing RNAs. There is, however, a third way to induce RNAi, and these are so called Dicer-substrate RNAs. Their potential utility for gene silencing was recognised following the finding by the Cleary/Hannon [Siolas et al. (2005) Nat. Biotechnol. 23:181] and Rossi [Kim et al. (2005) Nat. Biotechnol. 23: 222] groups that longer RNA duplexes of around 25-27 bp that undergo initial processing by the RNAi enzyme Dicer to yield the active small RNA are in some cases more efficient in gene than the equivalent siRNA. This is thought to be the consequence of Dicer actually forming part of the RiSC loading complex, thereby ensuring efficient hand-off of the small RNA product into the RiSC silencing complex.

There are a number of reasons why Dicer-substrate RNAs have not become a mainstream tool for inducing RNAi yet. Among these were the difficulty of manufacturing Dicer-substrate RNAs that would yield predictable small RNA effectors, non-specific perturbations of gene expression due to cytokine induction by the dsRNA, and the lack of reliable Dicer-substrate RNA design rules. Finally, RNAi inducers upstream of siRNAs may compete with more elements of the microRNA pathway than necessary and the longer length of RNAs will add to the cost and complexity of synthesis.

Some of these challenges, however, are being met mostly as a result of a collaboration between the Rossi lab of the City of Hope, California, and the nucleic acid synthesis company IDT which licensed Dicer-substrate RNAs for use in non-therapeutic applications. Creating dsRNA with one blunt end that contains DNA nucleotides on one strand and 2 nucleotide 3’ overhangs on the other end introduced directionality into Dicer processing. Moreover, it appears that the same modifications that can be introduced into standard siRNAs to avoid cytokine induction also work well for Dicer-substrate RNAs. One weakness that remains, however, is the lack of efficient Dicer-substrate RNA design rules. However, in collaboration with Bio-Rad, IDT is screening and developing sets of validated Dicer-substrate RNAs that have greater than 85% knockdown efficiencies.

It remains to be seen how well accepted Dicer-substrate RNAs will eventually become. With IDT, possibly the world’s largest synthetic nucleic acids supplier for research purposes, behind the technology, Dicer-substrate RNAs should be able to reach most researchers in the field. It will therefore be their hands-on experience, publications showing the benefits of Dicer-substrate RNAs and word-of-mouth that will determine the success of Dicer-substrate RNAs. As siRNAs have shown, a biotechnology that works predictably does not need much advertisement. Some interest meanwhile is demonstrated by the fact that Novartis is apparently testing a small library of Dicer-substrate RNAs for target validation purposes, and Nastech Pharmaceutical Company has obtained an exclusive license from the City of Hope for developing Dicer-substrate RNAs as therapeutics against a handful of gene targets.

With regards to IP issues, I would expect that their therapeutic use would require some kind of licensing agreement from the beneficiaries of the Tuschl I and II patents partly because of the 2nt 3’ overhang structures and the fact that Dicer-substrate RNAs are the immediate precursors of siRNAs. A precedent for this kind of licensing agreement has been set before by Benitec, which in 2005 has taken a license from Alnylam for the “targeted gene silencing mediated by short interfering RNAs (siRNAs) generated from DNA constructs introduced into cells”.

Saturday, June 2, 2007

In the latest issue of Molecular Cell, two separate groups report in back-to-back featured papers the discovery of miR-34a as a major contributor to p53-mediated apoptosis (Raver-Shapira et al. DOI: 10.1016/j.molcel.2007.05.017; Chang et al. DOI: 10.1016/j.molcel.2007.05.010). It is increasingly becoming clear that microRNAs play key regulatory roles in cancer and these studies contribute to our understanding of the mechanistic role of individual microRNAs. This opens up the prospect to use microRNAs both as diagnostics and therapeutics in cancer care management. Additionally, p53 function is implicated in many forms of cancer and a better understanding of how this “guardian of the genome” acts is desirable further informing treatment strategies.

Both groups set out to discover p53-regulated microRNAs by comparing microRNA profiles of p53-containing with p53-deficient cell lines, and found that miR-34a is particularly responsive to the presence of p53. Identification of functional p53 binding sites then confirmed that p53 directly acts as a transcriptional regulator of miR-34a expression. Through microRNA-inhibition and overexpression strategies both groups finally come to the conclusion that the p53-dependent response to genotoxic (= carcinogenic) insult is significantly effected by miR-34a activation which in turn regulates a number of genes involved in programmed cell death (apoptosis) and cancer progression. Strikingly, a major importance of miR-34a in mediating this response is further suggested by the finding by Joshua Mendell’s group (Chang et al.) that in all the 15 pancreatic cancer cell lines tested, miR-34a was significantly downregulated or even deleted.

These findings have important implications for the development of microRNAs both as diagnostics and cancer therapeutics. As such, the status of miR-34a could be used to predict the response of a tumour to a chemotherapeutic agent. miR-34a may also be used as part of microRNA diagnostics measuring a number of microRNAs for cancer screening purposes. Profiles showing low miR-34a may be indicative of the presence of a tumour. Maybe most intriguing would be the delivery of miR-34a mimics, either as synthetic siRNAs or in expressed form, to promote the apoptosis of cancer cells. Tt remains to be seen, however, which cancer types respond best to such a strategy and different degrees of efficiencies were reported in the different model systems used in the two papers.

A handful of companies may be interested in converting these ideas into real products. Rosetta Genomics (ticker: ROSG), a microRNA company of Israel, is certainly one of them and was heavily involved in the Shapira-Raver et al. studies. This company is founded on the discovery of proprietary microRNAs and have further licensed other microRNAs for diagnostic purposes from other institutions, most notably the Max-Planck Institutes of Germany, the Rockefeller and the MIT. It is not clear to me, however, who claims the rights to miR-34a, possibly the MIT given that David Bartel’s group first reported miR-34a following bioinformatic predictions (Lim et al. Science 299: 1540). Among other projects, Rosetta is currently developing microRNA diagnostics for Cancers of Unknown Primary (CUP) and pancreatic cancer diagnostics, the latter in collaboration with Asuragen, a privately held company of Austin, Texas. As these in-licensed microRNAs, however, are for diagnostic purposes only, it is well possible that other RNAi companies will get involved in miR-34a-based RNAi Therapeutics, as miR-34a would essentially be delivered in the form of RNAi effectors. David Bartel and the MIT e.g. have a close relationship with Alnylam (ticker: ALNY), which in turn owns many of the fundamental RNAi patents and therapeutic rights to many important microRNAs discovered by their scientific co-founder Thomas Tuschl of Rockefeller University.

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